Apparatus for the automatic determination of the coagulation, aggregation and or flocculation, or the like, rates of fluids, and novel reaction intensifying agent for use therewith

ABSTRACT

Apparatus for the determination of the coagulation rate or the like of fluids, wherein dispensing means are operable to successively dispense blood plasma samples, in turn and at a precisely metered rate, onto spaced-apart measured quantities or spots of a dried suspension of a reaction intensifying agent carried on an advancing film strip. The intensifying agent may comprise magnetic iron oxide particles. Subsequent to incubation of the sample-intensifying agent mixture, additional dispensing means are operable to dispense a precisely metered quantity of prothrombin reagent into the film strip. The resulting samplereagent-intensifying agent mixture is subjected to rotating magnetic fields, with the result that the moving magnetic particles promote mixing and, also, are operative to collect one or more of the fibrin strands, whereby the mixture, which is substantially turbid, undergoes a sharp change in its optical transmission properties, which change is detected by light sensitive means for the measurement of the prothrombin time of the blood plasma sample of interest.

Ilite atet Adler [72] Inventor: Stanford L. Adler, Monsey, NY.

[73] Assign ee: Technicon Corporation, Tarrytown, NY.

[22] Filed: Dec.'4, 1969 [21] Appl. No.: 881,971

[52] US. Cl. ..23/253 R, 23/183, 23/200, 23/230 B, 23/253 A, 23/253 TP,23/259, 73/64.l, 210/42, 210/222, 260/695, 356/39, 356/208 3,492,0961/1970 Hattersley ..23/230B OTHER PUBLICATIONS Hyland, Clotek SystemInstruction Manual, D- 219/35- OOSSB, 4 pages (Oct. 1968).

Primary Examiner-Joseph Scovronek Att0rneyS. P. Tedesco [5 7] ABSTRACTApparatus for the determination of the coagulation rate or the like offluids, wherein dispensing means are operable to suecessively dispenseblood plasma samples, in turn and at a precisely metered rate, ontospaced-apart measured quantities or spots of a dried suspension of areaction intensifying agent carried on an advancing film strip. Theintensifying agent may comprise magnetic iron oxide particles.Subsequent to incubation of the sample-intensifying agent mixture,additional dispensing means are operable to dispense a precisely metered quantity of prothrombin reagent into the film strip. The resultingsample-reagent-intensifying agent mixture is subjected to rotatingmagnetic fields, with the result that the moving magnetic particlespromote mixing and, also, are operative to collect one or more of thefibrin strands, whereby the mixture, which is substantially turbid,undergoes a sharp change in its optical transmission properties, whichchange is detected by 1i ght sensitive means for the measurement of theprothrombin time of the blood plasma sample of interest.

11 Claims, 5 Drawing Figures Patented March 21, 1972 2 Sheets-Sheet l TIIIIIIIIIIIIIIIIIIIII 1 EEEEQQE I I I I 1 I I 1 I I I IJ INVENTOR H. TimmEE.

.Patentecl March 21, 1972 3,650,698

2 Sheets-Sheet 2 FIG.42

. INVENTOR ST NFORD LER BY 1 ATTORNEY APPARATUS FOR Til-IE AUTOMATICDETERMINATION OF THE COAGULATION, AGGREGATION AND OR FLOCCULATION, ORTHE LIKE, RATES OF FLUIDS, AND NOVEL REACTION INTENSIFYING AGENT FOR USETHEREWITH BACKGROUND OF THE INVENTION the Prothrombin Time of bloodplasma samples.

2. Description of the Prior Art.

Although a wide variety of methods and apparatus are known in the priorart for the determination of the Prothrombin Time of blood plasmasamples for essential diagnostic use, it may be understood that, ingeneral, no prior art method and/or apparatus is known which canaccomplish this essential Prothrombin Time determination in fullyautomatic, rapid and consistently highly accurate manner with minimumutilization of the expensive thromboplastin reagent required therefor.

More specifically, and considering first the plurality of substantiallymanual classical Prothrombin Time determination methods in the nature ofthe visually monitored loop or tilt method as developed by Dr. A. J.Quick, and described in detail, for example, in the informativepublication Coagulation Procedures" published by Dade Reagents Inc, ofMiami, Florida, in January 1966, it is believed that the significantdisadvantages thereof in the areas of undue time consumption, inaccuracyas occasioned by unclear coagulation reaction endpoint indication,technician error, incompetence, and/or general inattention, and/orunduly high use of the required, expensive thromboplastin reagent, areso well known to those skilled in this art as to require no elaborationhere.

On the other hand, although a wide variety of automatic orsemi-automatic apparatus have been developed for Prothrombin Timedetermination, and are operable on such divergent bases as the detectionof changes in the viscosity of the blood plasma sample-thromboplastinreagent mixture, or the creation of an electrically conductive path bythe clotted materials, to indicate the end point or clotting time, thesesemi-automatic or automatic prior art apparatus will generally be foundto be incapable of consistently and rapidly providing accurateProthrombin Time determination results on a fully automatic basis withminimum use of the expensive thromboplastin reagent.

Probably the most widespread single source of error with re gard to theoperation of all of the Prothrombin Time determining methods andapparatus of the prior art is the fact that the same do not provide forsharp and dramatic change in a characteristic or property of the bloodplasma sample-thromboplastin reagent mixture at the end point of thecoagulation reaction which can be readily discerned and/or automaticallydetected to clearly indicate the precise point in time at which said endpoint occurs, and this disadvantage may be understood to be due in largemeasure to the fact that there are no known means by which said reactioncan be intensified or enhanced to provide this most desirable sharp anddramatic, readily discernible or automatically detectable change.

Of further disadvantage with regard to the Prothrombin Time determiningmethods and apparatus of the prior art is believed the fact that theabove-discussed disadvantages thereof generally become even morepronounced when the same are utilized with diluted blood plasma samples,to thereby render the same generally unsatisfactory for diag nostic usein establishing controls for essential anti-coagulant dosageadjustments.

OBJECTS OF THE INVENTION It is, accordingly, an object of this inventionto provide new and improved apparatus for the rapid, automatic, andconsistently accurate determination of the coagulation, aggregation orflocculation or the like rates of fluids, and which is particularlyadapted to such determination of the Prothrombin Time of blood plasmasamples.

Another object of this invention is the provision of apparatus as abovewhich operate to substantially minimize the amount of the expensivethromboplastin reagent required for a blood plasma sample ProthrombinTime determination to thus materially reduce the cost of suchdetermination.

Another object of this invention is the provision of a novel coagulationreaction intensifying or enhancing agent which provides for sharp anddramatic change in a readily discernible and/or automatically detectablecharacteristic of the blood plasma sample-thromboplastin reagent mixtureat the end point of the coagulation reaction,

Another object of this invention is the provision of apparatus as abovewhich are particularly adapted for operation with undiluted blood plasmasamples.

A further object of this invention is the provision of apparatus asabove which are fully and continuously automatic in operation andprovide a readily interpretable, permanent readout of the determinedblood plasma sample Prothrombin Times to thus eliminate most majorsources of technicianoccasioned inaccuracy.

A still further object of this invention is the provision of apparatusas above which require the use of only readily available components ofproven dependability in the fabrication thereof to thus insure longperiods of satisfactory, substantially maintenance-free apparatusoperation.

SUMMARY OF THE INVENTION As disclosed herein in a preferred embodimentdirected toward the determination of the Prothrombin Time of a pluralityof blood plasma samples, the apparatus of my invention comprise samplesupply means which supply a stream consisting of successive ones of saidblood plasma samples, and blood plasma sample dispensing means which areoperable to successively dispense said blood plasma samples at aprecisely metered rate. A novel coagulation reaction intensifying orenhancing agent which comprises opaque magnetic iron oxide particles asthe essential ingredient thereof is supplied in the form of spaced,measured quantities or spots of a dried suspension of said agent asdisposed on suitable carrier means which take the form of asubstantially transparent film strip.

Automatically operable thromboplastin reagent dispensing means areprovided and function to dispense precisely metered minimum quantitiesof said thromboplastin reagent upon demand. Further included aretemperature controlled means to effect blood plasma sample incubationand bring the same to appropriate coagulation reaction temperature,rotating magnetic field generation means to generate a rotating magneticfield through said temperature controlled means, and light sensitivedetection means to automatically detect the end point of the coagulationreaction and provide the desired Prothrombin Time determinationaccordingly.

In operation, said film strip is advanced to a first position on saidtemperature controlled means whereat a precisely measured quantity of ablood plasma sample is added to said reaction intensifying or enhancingagent to rapidly re-suspend the latter with the effect of said rotatingmagnetic field being to promote initial blood plasma sample-intensifyingor enhancing agent mixing and render the resultant mixture substantiallyturbid or opaque. After the expiration of a period of time predeterminedto provide for substantial blood plasma sample incubation, the filmstrip is advanced to said reagent dispensing means whereat a preciselymetered quantity of the thromboplastin reagent is added to thesubstantially turbid or opaque blood plasma sample-intensifying orenhancing agent mixture to commence the coagulation reaction with theeffect of said magnetic field on said magnetic iron oxide agent furtherpromoting thorough blood plasma sample-thromboplastin reagent mixing.Therefrom, the film strip is advanced to the operation position of saidlight sensitive detecting means, which position generally coincides withthe center of said rotating magnetic field. Thereafter, as the end pointof the coagulation reaction is reached, the substantially turbid oropaque blood plasma sample-thromboplastin reagent-intensifying agentmixture will undergo a sharp and dramatic change in optical propertythrough the collection of the rotating magnetic iron oxide particles inone or more of the fibrin strands generally centrally of the mixture tothus render the latter substantially transparent for immediate detectionby said light sensitive detecting means, and attendant provision ofaprinted readout of the Prothrombin Time of the blood plasma sample ofinterest. Operation of the apparatus is continuous in the mannerdescribed until each of the blood plasma samples of said successivestream thereof has been determined.

DESCRIPTION OF THE DRAWINGS The above and other objects and significantadvantages of my invention are believed made clear by the followingdetailed description thereof taken in conjunction with the accompanyingdrawings wherein:

FIG. 1 is a flow diagram depicting the new and improved apparatus of myinvention;

FIG. 2 is a top plan view ofa portion of the film strip carrier of theapparatus of FIG. 1 illustrating the disposition of the measuredquantities or spots of the reaction intensifying agent thereon;

FIG. 3 is a top plan view ofa portion of the film strip carrier of FIG.2 illustrating the manner in which the reaction intensifying agentpromotes blood plasma sample-thromboplastin mixing; and

FIGS. 4 and 5 illustrate the sharp and dramatic change in the turbidityof the blood plasma sample-reagent-reaction intensifying agent mixtureattendant the completion of the coagulation reaction.

DETAILED DESCRIPTION OF THE INVENTION Referring now to FIG. 1, samplesupply means which may, for example, take the general form of thoseshown and described in U.S. Pat. 3,134,263 issued May 26, I964 to EdwardB. M. DeJong, are indicated generally at and comprise a turntable 12upon which is disposed a circular array of blood plasma samplecontainers 14. A sample off-take device is indicated at 16 and comprisesa sample off-take probe 18 and probe operating means 20, respectively. Awash liquid receptacle 22 is disposed as shown adjacent the turntable12, while sample supply device drive means are indicated at 24 and areoperative to drive each of the turntable l2 and the sample off-takeprobe operating means in the manner described directly hereinbelow asindicated by the dashed lines extending therebetween.

In operation, the turntable I2 is intermittently rotated, or indexed, topresent each of the blood plasma sample containers 14 in turn to thesample off-take probe 18, while the latter is in turn intermittentlyoperated to immerse the inlet end ofthe off-take probe 18 in a thuslypresented sample container for a predetermined period of time toaspirate (as described in detail hereinbelow) a predetermined measuredvolume ofthe blood plasma sample therefrom, to then transfer the saidoff-take probe inlet end through the ambient air for immersion in thewash liquid receptacle 22 for a predetermined period of time to thusaspirate a predetermined measured volume of ambient air followed by apredetermined measured volume of said wash liquid therethrough, and tothen again transfer the said off-take probe inlet end through theambient air for immersion in the next presented sample container 14 fora predetermined period of time to thus aspirate another predeterminedmeasured volume of ambient air therethrough and commence the aspirationofa predetermined measured volume of the blood plasma sample from saidnext presented sample container.

As a result, it may be understood that a fluid stream consisting ofsuccessive, predetermined measured volumes of said blood plasma samplesas spaced, in each instance, by a segment of air, a segment of the washliquid from wash liquid receptacle 22, and a segment of air,respectively, will be supplied to the sample off-take probe 18.

A compressible tube or peristaltic proportioning pump which, forexample, may take the general form of that shown and described in U.S.Pat. 3,227,091 issued Jan. 4, 1966 to Jack Isreeli, et al., is indicatedgenerally in dashed lines at 26 and, as utilized herein, may beunderstood to comprise a plurality of resilient, compressible pump tubes28, 30, 32 and 34, respectively, each of which is progressivelycompressible or occludable by a plurality of non-illustrated pumprollers, in synchronous manner, to pump fluids therethrough in thedirection from right to left as indicated by the flow directional arrowsin FIG. 1.

The inlet end of compressible pump tube 30 is connected as shown to theoutlet end of the blood plasma sample off-take probe 18 to provide forthe aspiration of the blood plasma sample-wash liquid-air streamtherethrough as discussed hereinabove. The outlet end of compressiblepump tube 30 is connected as shown to one inlet ofajunction conduit 36,and the outlet of the latter is connected to a blood plasma samplesupply conduit 38.

The inlet end of compressible pump tube 28 is left open as shown toatmosphere, while the outlet end thereof is connected to one inlet ofthree-way valve means 40. One outlet conduit 42 of the three-way valvemeans 40 extends as indicated to atmosphere, while the other outletconduit 44 of said three-way valve means is connected to the other inletof the junction conduit 36. Accordingly is believed made clear that withthe three-way valve means 40 in a first operating position thereoftoconnect compressible pump tube 28 to outlet conduit 42, air will simplybe pumped through the said valve for return to atmosphere while, withsaid three-way valve means in a second position thereof to connectcompressible pump tube 28 to valve outlet conduit 44, air will be pumpedto junction conduit 36 for merger with the blood plasma sample-washliquid-air stream being pumped therethrough from compressible pump tube30.

A reagent container is indicated at 46 and, for use as disclosed hereinin determining the end point of the blood coagu lation reaction, orProthrombin Time, of the blood plasma samples from sample supply means10, may be understood to contain a suitable supply of thromboplastinreagent as indicated at 48. A reagent supply conduit 50 is disposed asshown within the reagent supply container 46 so that the inlet end ofthe former is in close proximity to the bottom of the latter for obviouspurpose, and the outlet end of reagent supply conduit 50 is connected asshown to the inlet end of compressible pump tube 32, whereby is believedmade clear that operation of the peristaltic pump 26 will result in thepumping of the reagent 48 from said supply container through saidcompressible pump tube in the indicated direction.

The outlet end of compressible pump tube 32 is connected as shown to oneinlet ofajunction conduit 52, while the outlet of said junction conduitis connected to a reagent dispensing conduit 54. A reagent by-pass andreturn conduit is indicated at 56, and the inlet end thereof isconnected as shown to the other outlet of the junction conduit 52, whilethe outlet end of said reagent by-pass and return conduit extends asshown into the reagent supply container 46 for the return of thethromboplastin reagent thereto as described in detail hereinbelow.

Temperature control means which include a heating coil 58 connected asshown in the reagent supply conduit 54 adjacent the outlet end of thelatter, and a temperature control bath as indicated in dashed lines at60, are included to enable the precise control of the temperature of thethromboplastin reagent immediately prior to the dispensing thereof fromsaid conduit outlet end as described in detail hereinbelow.

A suction conduit for reagent return is indicated at 62 and is connectedas shown to the reagent supply conduit 54 immediately adjacent theoutlet end of the latter. The outlet end of the suction conduit 62 isconnected as shown to the inlet end of compressible pump tube 34, whilethe outlet end of the latter is connected to the inlet end of a reagentreturn conduit 64 which extends as shown to within the reagent supplycontainer 46 to providefor the return of the thromboplastin reagentthereto, against as described in detail hereinbelow.

A supply roll of a strip of a film substrate 66 which is made from anymaterial having suitable strength, light transmission, and chemicalinertness characteristics in the nature, for examshown on rotatablesupport means as indicated at 68.. As best seen in FIG. 2, measuredquantities or spots 70 of a dried suspension of a reaction intensifyingor enhancing agent are disposed at substantially equally spacedintervals on the upper surface of the film strip 66. For use asdisclosed herein the automatic, sequential determination of therespective Prothrombin Times of a plurality of blood plasma samples,said reaction intensifying agent may be understood to comprisesubstantially opaque particles of a paramagnetic material such asmagnetic iron oxide particles of the nature used in the manufacturetape, as homogeneously suspended before drying in a suitable carriersolution. Magnetic iron oxide particles of this nature are of generallyacicular configuration and have a major dimension in the range of 0.40to 0.60 microns. Alternatively, such particles may be constituted bycobalt or nickel oxide particles.

More specifically, and for use as disclosed herein in intensifying orenhancing the blood plasma sample-thromboplastin reaction, said reactionintensifying agent may consist of a suspension of approximately 50 gramspercent magnetic iron oxide particles in approximately 25 percent PVPwhich contains approximately 5 percent glycerine, before drying, andsaid suspension may be applied to the film strip 66 for the formation ofthe spots 70 in any appropriate manner as, for example, through the useof well-known silk screening techniques. Preferably, the pH of thereagent intensifying or enhancing agent is adjusted, if necessary, toinsure that the same will not react with the thromboplastin reagent uponthe mixture thereof.

The particular PVP and glycerine constituents of the magnetic iron oxideparticle suspension, and the listed relative percentages of said ironoxide particles and said PVP and glycerine constituents in the reactionintensifying or enhancing agent, may be understood to be particularlyeffective in in suring the desired, substantially instantaneousre-suspension of said iron oxide'particles in an aqueous solution uponthe mixture thereof with the respective blood plasma samples asdescribed in detail hereinbelow. It is, however, believed clear thatother and different suspensions of said magnetic iron oxide particles inother and different relative percentaged with regard to said PVP andglycerine constituents, and/or with other and different suspendingagents, may prove equally useful.

Boundary means in the nature of those indicated at 72 in F1 2 willpreferably be formed on the film strip 66 in any appropriate manner as,for example, through embossing of the latter, to effect the substantialcontainment in the indicated area of the reaction intensifying orenhancing agent and the respective blood plasma samples upon there-suspension of the former in the latter as described in detailhereinbelow.

Heat sink means having a substantially level, flat upper surface areindicated at 74, and heating coil means 76 are disposed as shownadjacent the lower surface of said heat sink means to provide for thesubstantial maintenance of the latter at a desired temperature as shouldbe obvious. A mirror 78 is disposed as shown in the heat sink means 74in such manner that the upper surface of said mirror is substantiallyflush with the upper surface of said heat sink means.

Film strip guide and drive means are provided and comprise an idlerroller 80, a film strip advance drive roller 82, and a film strippressure roller 84 cooperatively associated with the latter as shown.Accordingly, intermittent driven rotation of from the film strip supplyroll, the passage thereof beneath idler roller 80, the passage thereofover the upper surface of heat sink means 74 in close contact therewith,and the sub- 88 and may be seen to comprise a suitable, generallycylindrical opaque housing 90 having a focusing lens 92 disposed thereinas shown. A light source is indicated at 941, and light detection meanswhich may, for example, take the form of a photoelectric cell, areindicated at 96, and each of said light source and said photoelectriccell are positioned within the housing 90 as shown in such manner thatthe light beam will be focused as indicated by the focusing lens 92 forimpingement upon the mirror 78 and reflection therefrom for refocusingby the focusing lens 92 for impingement upon the active surface of thephotoelectric cell 96.

Reaction timer and readout printing means are indicated schematically at98 and are operatively associated as shown with the photoelectric cell96 to provide for reaction timing and readout printing on a tape 99 asdescribed in detail hereinbelow.

A bar magnet of appropriate field strength is indicated at 100 and issupported as shown for rotation at appropriate rate through operation ofdrive motor means 102 to thereby establish a rotating magnetic field,the lines of force of which will, of course, extend through and abovethe heat sink means 74. Preferably, the bar magnetic 100 is disposedrelative to the mirror 78 in such manner that the respective centersthereof are in substantial vertical alignment as seen in FIG. 1, wherebyit may be understood that the center of the rotating magnetic fieldestablished by rotation of the bar magnet 100 will be generallycoincident with the center of the mirror 78.

Multi-element snap-action valve means are indicated generally in dashedlines at 104 and may, for example, take the general form of those shownand described in the co-pend ing application for United States Patent ofCarl V, Johnson et al., entitled New and Improved Method and Apparatusfor Simultaneously Controlling the Flow of Fluids in a Plurality of FlowPaths, Ser. No. 864,262, filed Sept. 22, 1969 and assigned to theassignee hereof. Each of the reagent by-pass and return conduit 56, thereagent dispensing conduit 54, and the suction conduit 62 pass as shownthrough the valve means 104 for control by the latter of the respectivefluid flows therethrough and, to this effect, it may be understood thatat least the portion of each of said conduits which passes through saidvalve means is constituted by a compressible tube or conduit asindicated respectively at 106, 108 and 110.

The valve means 104 comprise a valve actuator member 111 havingprojection bar members 112, 114, and 116 formed thereon with said barmembers being respectively operatively associated with the compressibleconduit portion 106, and 110. In operation for use as disclosed indetail hereinbelow, it may be understood that the valve means 104 arearranged so that the valve actuator 111 is pivotally moveable, inextremely rapid, snap-action manner between a first position thereofwherein bar member 114 contacts and substantially compresses or occludescompressible conduit portion 108 against a non-illustrated platen tosubstantially prevent fluid flow therethrough, while neither of barmembers 112 or 116 contacts compressible conduit portions 106 and andthus does not interfere with the respective flow of fluids therethrough,to a second position of said valve actuator wherein bar members 112 and116 respectively contact and substantially compress or occludecompressible conduit portions 106 and 110 against said platen tosubstantially prevent fluid flow therethrough, while bar member 114 doesnot contact compressible tube portion 108 and thus does not interferewith the flow of fluids therethrough. Accordingly, it is believed madeclear that with said valve actuator 111 in said first position thereof,fluid flow through reagent dispensing conduit 54 will be substantiallyprevented. while fluid flow through the respective reagent by-pass andreturn conduits 56 and suction conduit 64 will be unaffected.Conversely, with said valve actuator 111 in said second positionthereof, it is believed clear that fluid flow through the respectivereagent by-pass and return conduit 56 and suction conduit 64 will besubstantially prevented, while fluid flow through reagent dispensingconduit 54 will be unaffected.

A blood plasma sample dispensing probe is indicated at 120 and the inletend thereof is connect-ed as shown to the outlet end of blood samplesupply conduit 38. The blood sample dispensing probe 120 is supported asshown adjacent the outlet end thereof from the arm 122 of blood sampledispensing probe operating means 124. The blood sample probe operatingmeans 124 are operated from drive motor means as indicated at 126A foroscillatory movement to move the inlet end of the blood sampledispensing probe 120 in an arc between the depicted first positionthereof wherein said outlet end is in substantial vertical alignmentwith a measured quantity or spot 70 of the reagent intensifying orenhancing agent on the upper surface of film strip 66 when the latter isin a specific position thereof as described in detail hereinbelow, and asecond position of the probe as depicted in phantom in FIG. 1 whereinsaid dispensing probe outlet end is in general vertical alignment with ablood plasma sample and wash liquid collection receptacle 126A whichleads as indicated to waste.

Programmer means which may take any appropriate form in the nature, forexample, of cam-operated electrical programmer means are indicatedschematically at 128 and are operatively connected as indicated by thedashed lines to each of the sample supply means drive motor 24, thethree-way valve means 40, the multi-element valve means 104, the bloodplasma sample dispensing probe operating means drive motor 126, the filmstrip advance drive roller drive motor 86, the photoelectric cell 96,and the timer and readout printing means 98, respectively.

OPERATION For typical use in the automatic, sequential determination ofthe respective Prothrombin Times of a plurality of blood plasma samples,it may be understood that each of the blood plasma sample containers 14would contain an undiluted blood plasma sample from a different patient,each of which will have been previously treated in classical manner witha fixative reagent in the nature of sodium oxalate or sodium citrateimmediately upon withdrawal from the patient, and will preferably bemaintained at approximately 4 to 5 C. in the relevant blood plasmasample container 14 to inhibit clotting factor deterioration through theuse of non-illustrated cooling means which may be included in oroperatively associated with the sample supply device 10.

The three-way valve means 40 would be arranged to operate, under thecontrol of programmer 128, to be in the second position thereof tointroduce air to the blood plasma sample-wash liquid-air stream flowingthrough junction conduit 36 only when the wash liquid segments areflowing therethrough to thus air segment each of said wash liquidsegments to materially improve the cleansing action thereof, whileinsuring the flow of each of the aspirated blood plasma samples as acontinuous stream to the blood plasma sample dispensing probe 120. Thereagent supply container 46 would, of course, contain a supply of thethromoplastin reagent 48 sufficient for the Prothrombin Timedetermination of each of the blood plasma samples carried from thesample supply means 10.

In addition, the film strip advance drive roller motor 86 would bearranged to operate the film strip advance drive roller 82, under thecontrol of programmer 128, to alternatively, intermittently advance thefilm strip 66 to two positions thereof relative to the focal point oflens 92, the outlet end of reagent dispensing conduit 54, and the outletend ofthe blood plasma sample dispensing probe 120, respectively, and toena ble the dwelling of said film strip in each of said positions fordifferent predetermined periods of time as described in detailhereinbelow. More specifically, it may be understood that said filmstrip advance drive roller would initially be operated to advance thefilm strip to the position thereof depicted in FIG. 1 wherein a leadingmeasured quantity or spot 70 of the reaction intensifying or enhancingagent (as now thoroughly mixed and re-suspended with a blood plasmasample and a precisely metered amount of the thromboplastin reagent inthe manner described in detail hereinbelow) would be disposed asindicated at position C on the mirror 78 at the exact focal point of thefocusing lens 92, while the next succeeding or trailing measuredquantity or spot 70 of said reaction intensifying or enhancing agentwould be disposed as indicated at position A directly below the outletend of blood plasma sample dispensing probe when the same is in thedepicted blood plasma sample dispensing position thereof as shown inFIG. 1. Accordingly it is believed made clear that the spacing 5 (FIG 2)between said measured quantities or spots 70 of the reactionintensifying or enhancing agent on the upper surface of the film strip66 is predetermined to substantially coincide with the distance betweensaid lens focal point on mirror 78 and the point on heat sink 74 whichis in substantial vertical alignment with the outlet end of the bloodplasma sample dispensing probe 120. In said second position to which thefilm strip 66 is intermittently advanced by operation of said film stripadvance drive roller 82, it may be understood that the former would bepositioned so that the measured quantity or spot 70 of the reactionintensifying or enhancing agent which had been disposed at position Awould now be advanced for disposition in substantial vertical alignmentwith the outlet end of reagent dispensing conduit 54 at position B asindicated in FIG. 1, it being believed clear that with the film strip 66thusly disposed, no measured quantities or spots 70 of the reactionintensifying or enhancing agent will be disposed at either of positionsA or C.

The multi-element valve means 104 would be arranged to operate, againunder the control of programmer 128, to be in said second positionthereof to enable reagent flow from reagent supply conduit 32 throughcompressible conduit portion 108 to reagent dispensing conduit 54 onlyfor a period of time predetermined to substantially coincide with theperiod of time in which the film strip 66 is dwelled in a said secondposition thereof to position a measured quantity or spot 70 of thereaction intensifying or enhancing agent in substantial verticalalignment with the outlet end of said reagent dispensing conduit. At allother times, it may be understood that the multielement valve means 104would be arranged to be in said first position thereof to permit fiuidflow only through compressible conduit portions 106 and 110. Withfurther regard to the reagent dispensing conduit 54, it may beunderstood that temperature control bath 60 would be arranged tomaintain the temperature of the temperature control coil 58 atapproximately 37 C. to thus insure the dispensing of the thromboplastinreagent at substantially this temperature.

The blood plasma sample dispensing probe operating means 124 would bearranged to operate, through drive motor 126A under the control ofprogrammer 128, to position the blood plasma sample dispensing probe 120as depicted in the blood plasma sample dispensing position thereof onlyfor a part of the time when the film strip 66 is dwelled in a said firstposition thereof with a measured quantity or spot 70 of the reagentintensifying or enhancing agent being disposed as depicted in FIG. 1 atposition A, and to at all other times position said blood plasma sampledispensing probe in the position depicted in phantom in FIG. 1 wherebythe remainder of each of the blood plasma samples that is not dispensedfor test, and the respective inter-sample wash liquid segments, will bedispensed into receptacle 126 for flow therefrom to waste.

Finally, heating coil means 76 would be arranged to operate to maintainthe temperature of the heat sink means 74 at approximately 37 C.

In operation, and assuming steady state operational conditions to havebeen reached and the film strip 66 to have just been advanced to a firstposition thereof to dispose succeeding measured quantities or spots 70of the reagent intensifying or enhancing agent at positions A and C asdepicted in FIG. 1, it may be understood that the blood plasma sampledispensing probe 120 will just have been moved to the depicted positionthereof and that a blood plasma sample willjust be commencing to flowtherefrom as a continuous stream to fall upon and mix with the measuredquantity or spot 70 of the reaction intensifying or enhancing agent nowdisposed at position A with resultant almost instantaneous re-suspensionof the magnetic iron oxide particles therein. This almost instantaneousresuspension, and concomitant commencement of the thorough mixing ofsaid iron particles in said blood plasma sample will be significantlyenhanced by the effect of the rotating magnetic field generated by therotation of the bar magnet 100 upon said magnetic iron oxide particlesas should be obvious.

After the expiration of a time period predetermined to enable thedispensing of the desired amount of the blood plasma sample, which timeperiod may, for example, be of approximately to 2 seconds duration, itmay be understood that the blood plasma sample dispensing probeoperating means will be actuated to move said probe to said secondposition thereof directly over wash-liquid collection receptacle 126 toenable the flow of the remainder of the blood plasma sample, and thesucceeding air segment, air segmented wash liquid segments, and airsegments, through said blood plasma sample dispensing probe to insure athorough cleansing of the latter and prevent the contamination of thesucceeding blood plasma sample by the residue of the just dispensedblood plasma sample, to obvious advantage.

Preferably, the film strip advance drive roller 82 will, at this point,be maintained stationary for approximately 55 seconds to dwell the filmstrip 66 in this first position thereof for that period of time to bringthe thusly dispensed blood plasma sample and the measured quantity orspot 70 of the reaction intensifying or enhancing agent up to thepreferred test tempera ture of approximately 37C. and commence therequired incubation of said blood plasma sample.

At the expiration of this dwell period, it may be understood that thefilm strip advanced drive roller 82 will be operated to advance the filmstrip 66 to said second position thereof wherein the now thoroughlymixed blood plasma sample and reagent intensifying or enhancing agentmeasured quantity 70 will be disposed at position B directly beneath theoutlet end of reagent dispensing conduit 54. concomitantly, themultielement valve means 104 will be rapidly shifted to the secondposition thereof to enable the commencement of reagent dispensing, inprecisely metered amount, through the reagent dispensing conduit 54 atapproximately 37C. As the thusly dispense reagent joins the blood plasmasample and the now re-suspended magnetic iron oxide particles tocommence the desired blood plasma sample-thromboplastin reagentreaction, it may be understood that the advantageous thorough mixturethereof will continue to be enhanced by the action of the rotatingmagnetic field on said magnetic iron oxide particles. With a dwellperiod of approximately 2 seconds for the film strip 66 in this secondposition thereof, it may be understood that a reagent dispensing timeranging from approximately 1% to 2 seconds may be utilized. Too, forpurposes of determining the Prothrombin Time of the blood plasma sampleof interest, it may be understood that the time of shifting of themulti-element valve means to said second position thereof may be takenas time 0 and communicated from the programmer 128 to the timer andreadout printing the means 98 since this time substantially coincideswith the time the thromboplastin reagent first contacts said bloodplasma sample to commence the coagulation reaction.

At the expiration of the period of time predetermined to be sufficientto enable the precise dispensing of the desired quantity of thethromboplastin reagent through reagent dispensing conduit 54, it may beunderstood that multi-element valve means 104 will be rapidly shifted toreturn to said first position thereof wherein further reagent flow fromreagent supply conduit 32 to reagent dispensing conduit 54 will beprevented by the abrupt closure of compressible conduit portion 108, andthe supply of reagent being pumped through compressible pump tube 32by-passed instead through reagent by-pass and return conduit 56 forreturn to reagent supply container 46. This shifting of themulti-element valve means to said second position thereof will, inaddition, be effective to open compressible conduit portion withattendant creation of considerable suction in suction line 62 throughthe operation of compressible pump tube 34 to thus insure that anythromboplastin reagent remaining in the outlet end portion of thereagent dispensing conduit 54 after the completion of reagent dispensingis sucked therefrom through the suction 62 for return to reagent supplycontainer 46 on reagent return conduit 64 to thus further insure theprecise dispensing of exactly the desired amount, only, of said reagentand substantially prevent any waste of the latter to obviouslysignificant ad vantage.

At the expiration of this approximately 2 seconds dwell period, the filmstrip advance drive roller 82 is operated to again advance the filmstrip 66 to a said first position thereof wherein the blood plasmasample-reagent-reagent intensifying or enhancing agent mixture underdiscussion will be advanced to position C which, as discussedhereinabove, substantially coincides with the focal point of focusinglens 92 on mirror 78. Although appropriate spacing between therespective measured quantities or spots 70 of the reaction intensifyingor enhancing agent on the film strip 66 relative to the spacing betweensaid focusing lens focal point and the outlet end of blood plasma sampledispensing probe when the latter is in the dispensing position thereofmay be utilized to insure that the mixture of interest comes to restprecisely at position C, it may be understood that a further input fromthe photoelectric cell 96 to the programmer 128 may, in addition, beutilized to insure this occurs. More specifically, it may be understoodthat since this blood plasma sample-reagent-reaction intensifying agentmixture is at this point still substantially opaque or turbid due to thewide distribution of the substantially opaque magnetic iron oxideparticles therein, the movement of the said mixture into position C willfunction to break the beam of light from light source 94 to saidphotoelectric cell, and that this occurrence may be utilized for theprovision ofa control signal to programmer 128 to immediatelydiscontinue operation of the film strip advance drive roller drive motor86 as should be obvious.

As the blood plasma sample-reagent-reaction intensifying agent mixtureof interest assumes position C, it may be understood that the verythorough mixing thereof through movement of the magnetic iron oxideparticles under the influence of the magnetic field generated by therotating bar magnet 100 will now be very significantly enhanced. Morespecifically, and referring now to FIG. 3 wherein a number of theacicular magnetic particles are depicted to better illustrate thethoroughness of this mixing, it may be understood that each of saidparticles will be caused by the action of said magnetic field to bothrotate about its own axis and to rotate about the center of the mixture,as indicated in each instance by the rotational direction arrows, tosignificantly promote the desired blood plasma sample-thromboplastinreaction as should be obvious.

As the coagulation reaction proceeds it may be understood thatpolymerization of the fibrinogin in the blood sample into fibrin strandswill result, and that these fibrin strands will be in essence collectedby the multitude of rotating magnetic iron oxide particles which will becontinually rotated therethrough, As this fibrin strand collectioncontinues to occur, strand collection continues to occur, the saidfibrin strands will become interwoven until such time as the same are inessence collected by the magnetic iron oxide particles into one or morerelatively large globules or agglomerates thereof to indicate that thereaction end point or blood plasma sample clotting time has beenreached.

More specifically, and referring now to FIGS. 4 and 5, it may beunderstood that FIG. 4 depicts the blood plasma sample-reagent-reactionintensifying agent mixture as the same initially assumes position C andclearly illustrates that the said mixture is, at this point,substantially turbid or opaque due to the substantially evendistribution of the multitude of magnetic iron oxide particles therein.However, as the coagulation reaction progresses to the end point orclotting time thereof, as discussed directly hereinabove, it may beunderstood that the interaction between the fibrin strands and saidmagnetic iron oxide particles, and collection thereof in one or morerelatively large globules as indicated at 73 in FIG. 5 will result in arapid and dramatic change in the optical characteristics of the saidmixture to those depicted in FIG. 5, wherein the said fibrin-magneticiron oxide particle globules will be collected generally centrally ofsaid mixture to result in sharp and dramatic change in the lighttransmission properties thereof from the substantially turbid or opaqueto the substantially translucent or transparent.

This sharp and dramatic change in the turbidity or opaqueness ofthemixture will, of course, be immediately detected by the photoelectriccell 96 through the sharp and dramatic reopening of the light paththereto for the beam of light from light source 94, as should beobvious, with resultant instantaneous provision thereby of anappropriate signal to the timer and readout printing means 98 to stopsaid timer (it being recalled that said timer was started at time 0 bythe shifting of multi-element valve means 104 into said second positionthereof to commence thromboplastin reagent dispensing) and provide aprinted readout in seconds upon the tape 99 of the Prothrombin Time ofthe blood plasma sample of interest, to be followed by appropriateresetting of the timer and readout printing means 98 in preparation forthe determination of the Prothrombin Time ofthe succeeding blood plasmasample.

Concomitantly with the movement of the blood plasmasample-reagent-reaction intensifying agent mixture under discussion intoposition C, it is believed clear that the succeeding measured quantityor spot 70 of the reaction intensifying or enhancing agent or film strip66 will, of course, have been moved into position A for addition of thesucceeding blood plasma sample thereto through blood plasma sampledispensing probe 120, and commencement of the approximately 55 secondincubation period of said succeeding blood plasma sample.

Operation ofthe apparatus of my invention is, of course, automaticallycontinuous as described until the Prothrombin Time for each of the bloodplasma samples supplied from sample supply device has been determined.For a typical application of this nature, 60 of said blood plasmasamples may be positioned at one time on the sample supply meansturntable l2, and approximately only one hour will be required for thedetermination of the Prothrombin Times of all of said blood plasmasamples.

Of particular advantage with regard to the new and improved, automaticcoagulometer apparatus of my invention is believed the fact that, asapplied to the determination of the respective Prothrombin Times of aplurality of blood plasma samples, the same provides for absoluteminimization in the amount required of the expensive thromboplastinreagents. More specifically, it is believed clear that, through the useof the proportioning pump 26, the rapidly acting multi-element valvemeans 104, the reagent dispensing conduit 54, the reagent by-pass andreturn conduit 56, and the suction conduit 62, the precisely metereddispensing of only that quantity of the thromboplastin reagent which isrequired for the test purposes is insured, and that waste of saidreagent is substantially prevented. Thus, for example, the apparatus ofmy invention may be understood to make possible the precise andautomatic determination of the Prothrombin Time of an undiluted bloodplasma sample in a ratio of approximately 0.02 ml. of thromboplastinreagent to each 0.01 ml. of blood plasma sample as opposed, for example,to blood plasma sample Prothrombin Time determination made in accordancewith the principles of the prior art which require approximately 0.2 ml.of thromboplastin reagent per 0.1 ml. of blood plasma sample. Thus maybe readily appreciated that the apparatus of my invention make possiblea very substantial and significant reduction of approximately percent inthe amount of expensive thromboplastin reagent required per blood plasmasample Prothrombin Time determination.

The significant advantages attendant the fully automatic and extremelyaccurate operation of the apparatus of my invention with regard to theelimination of technician errors and the like are believed so clear asto not require further elaboration here.

Although disclosed hereinabove in the form of a preferred embodimentwhich is directed toward the automatic, successive determination of therespective Prothrombin Times of a plurality of blood plasma samples, itis believed clear that the apparatus of my invention would havesignificant utilization for different purposes. More specifically, it isbelieved clear that through suitable modification the said apparatuscould readily be adapted to the determination, for example, of thepartial thromboplastin time, or PTT, of the blood plasma samples toenable the use thereof for the more specific isolation of the factor orfactors causing deficiencies in the clotting time of a patient's blood.In addition, and again with suitable modification, it is believed clearthat the apparatus of my invention may be utilized in the determinationof the end point of a polymerization type reaction, the end point ofwhich is evidenced by an abrupt change in viscosity, in a wide varietyof liquids other and different than blood plasma samples. Thus, forexample, the apparatus of the invention could be utilized to determinethe end point of the flocculation reaction as would occur in pregnancytesting and/or the end point of the agglutination reaction as wouldoccur in testing for rheumatoid arthritis.

Too, although disclosed herein by way of example for the determinationof the Prothrombin Times of undiluted blood plasma samples, it may beunderstood that the apparatus of the invention is applicable for suchdetermination in reliable and accurate manner for blood plasma samplesdiluted down as far as 5 percent to thus enable the use of the apparatusto establish anti-coagulant dosage controls for essential therapeuticuse.

While I have shown and described the preferred embodiment of myinvention, it will be understood that the invention may be embodiedotherwise than as herein specifically illustrated or described, as thatcertain changes in the form and arrangement of parts and in the specificmanner of practicing the invention may be made without departing fromthe underlying idea or principles of this invention within the scope ofthe appended claims.

What is claimed is:

1. In apparatus for determining the coagulation rates or the like offluids through the measurement of the reaction time thereof with acoagulation reagent or the like, means to mix a said fluid and saidreagent with a reaction intensifying agent which, upon activation, willintensify said reaction and provide a readily detectable change in anoptical property of said fluid-reagent-reaction intensifying agentmixture at the completion of said reaction, said reaction intensifyingagent comprising a plurality of paramagnetic particles, means toactivate said reaction intensifying agent by subjecting said mixture toa moving magnetic field, so as to effect relative movement between saidparticles and said mixture, and means to monitor said optical propertyand to detect said optical property change to thus effect themeasurement of said reaction time.

2. Apparatus as in claim 1 wherein, said reaction intensifying agentparticles are of a material which is substantially chemically inert withregard to said fluid and said reagent, whereby said reactionintensifying agent will not interfere chemically with said reaction.

3. Apparatus as in claim 1 wherein, said activating means are operableto generate a rotating magnetic field, whereby said reactionintensifying agent particles will be rotated through said mixture underthe influence thereof. 7

4. Apparatus as in claim 3 wherein, said reaction intensifying agent issubstantially opaque and generally uniformly distributed throughout saidfluid-reagent-reaction intensifying agent mixture at the commencement ofsaid reaction to render the same substantially turbid, and said reactionintensifying agent is operable to collect generally centrally of saidfluid-reagent-reaction intensifying agent mixture under the influence ofsaid activating means only at the completion of said reaction withconcomitant change in the turbidity of said fluid-reagent-reactionintensifying agent mixture.

5. Apparatus as in claim 4 wherein, said means to mix a said fluid andsaid reagent with said reaction intensifying agent comprise, means tosuccessively supply predetermined quantities of said reactionintensifying agent, means to successively mix a predetermined quantityof a said fluid with each of said reaction intensifying agentquantities, and means to succes-. sively mix a predetermined minimumquantity of said reagent with each of said reaction intensifyingagent-fluid quantity mixtures to effect said reaction.

6. Apparatus as in claim 5 wherein, said means to successively supplysaid predetermined quantities of said reaction intensifying agentcomprise, strip-like carrier means having said quantities disposedthereon in substantially equally spaced manner.

7. Apparatus as in claim 6 wherein, said carrier means are substantiallytransparent, and said detecting means comprise light sensitive meanswhich are operable to direct a beam of light through said mixture andthrough said carrier means to thereby detect said change in theturbidity of said fluid-reagent-reaction intensifying agent mixturethrough the detection of the attendant change in the light transmissioncharacteristics thereof.

8. Apparatus as in claim 6 wherein, said magnetic field generation meansare disposed remotely of said carrier means and said mixtures.

9. Apparatus as in claim 1 wherein, said means to mix a said fluid andsaid reagent with said reaction intensifying agent comprise, means tosuccessively supply predetermined quantities of said reactionintensifying agent, means to successively mix a predetermined quantityof a said fluid with each of said reaction intensifying agentquantities, and means to successively mix a predetermined minimumquantity of said reagent with each of said reaction intensifyingagent-fluid quantity mixtures to effect said reaction.

10. Apparatus as in claim 9 wherein, said means to succes sively supplysaid predetermined quantities of said means reaction intensifying agentcomprise, strip-like carrier means having said quantities disposedthereon in substantially equally spaced manner.

11. Apparatus as in claim 10 wherein, said carrier means aresubstantially transparent.

2. Apparatus as in claim 1 wherein, said reaction intensifying agentparticles are of a material which is substantially chemically inert withregard to said fluid and said reagent, whereby said reactionintensifying agent will not interfere chemically with said reaction. 3.Apparatus as in claim 1 wherein, said activating means are operable togenerate a rotating magnetic field, whereby said reaction intensifyingagent particles will be rotated through said mixture under the influencethereof.
 4. Apparatus as in claim 3 wherein, said reaction intensifyingagent is substantially opaque and generally uniformly distributedthroughout said fluid-reagent-reaction intensifying agent mixture at thecommencement of said reaction to render the same substantially turbid,and said reaction intensifying agent is operable to collect generallycentrally of said fluid-reagent-reaction intensifying agent mixtureunder the influence of said activating means only at the completion ofsaid reaction with concomitant change in the turbidity of saidfluid-reagent-reaction intensifying agent mixture.
 5. Apparatus as inclaim 4 wherein, said means to mix a said fluid and said reagent withsaid reaction intensifying agent comprise, means to successively supplypredetermined quantities of said reaction intensifying agent, means tosuccessively mix a predetermined quantity of a said fluid with each ofsaid reaction intensifying agent quantities, and means to successivelymix a predetermined minimum quantity of said reagent with each of saidreaction intensifying agent-fluid quantity mixtures to effect saidreaction.
 6. Apparatus as in claim 5 wherein, said means to successivelysupply said predetermined quantities of said reaction intensifying agentcomprise, strip-like carrier means having said quantities disposedthereon in substantially equally spaced manner.
 7. Apparatus as in claim6 wherein, said carrier means are substantially transparent, and saiddetecting means comprise light sensitive means which are operable todirect a beam of light through said mixture and through said carriermeans to thereby detect said change in the turbidity of saidfluid-reagent-reaction intensifying agent mixture through the detectionof the attendant change in the light transmission characteristicsthereof.
 8. Apparatus as in claim 6 wherein, said magnetic fieldgeneration means are disposed remotely of said carrier means and saidmixtures.
 9. Apparatus as in claim 1 wherein, said means to mix a saidfluid and said reagent with said reaction intensifying agent comprise,means to successively supply predetermined quantities of said reactionintensifying agent, means to successively mix a predetermined quantityof a said fluid with each of said reaction intensifying agentquantities, and means to successively mix a predetermined minimumquantity of said reagent with each of said reaction intensifyingagent-fluid quantity mixtures to effect said reaction.
 10. Apparatus asin claim 9 wherein, said means to successively supply said predeterminedquantities of said means reaction intensifying agent comprise,strip-like carrier means having said quantities disposed thereon insubstantially equally spaced manner.
 11. Apparatus as in claim 10wherein, said carrier means are substantially transparent.